High yield process for in vitro production of serum-free and mitogen-free interleukin-2

ABSTRACT

An improved in vitro cell culture process for producing in high yield and purity a serum-free and mitogen-free interleukin-2-containing conditioned supernatant. The incubation steps during stimulation and conditioning of the interleukin-2 producing cells is carried out with a rapidly rotating roller bottle culture system. Yields of interleukin-2 are on the order of 10-fold and higher than obtained by similar cultivations carried out in flat dishes or tubes or in a roller bottle culture system rotated at conventional speeds. Improvement in yields in both the static and roller culture systems are also achieved by incubating the IL-2 producing cells under high oxygen concentrations, for example atmospheres of at least 70% O 2  and at least 5% CO 2 . In addition to using peripheral mononuclear blood cells (PBL) as the source of leukocytes containing the IL-2 producer cells, it is also possible to use buffy coat cells which are readily available as a waste by-product from blood banks.

This is a continuation-in-part of our prior copending application Ser.No. 247,769, filed Mar. 26, 1981, now U.S. Pat. No. 4,390,623 issued onJune 28, 1983, which in turn is a continuation-in-part of applicationSer. No. 193,112, filed Oct. 2, 1980, and now abandoned.

This invention relates to an improved method for producing T-cell growthfactor (TCGF), also known as Interleukin-2 (IL-2) in an in vitro culturesystem. More particularly, this invention relates to improvements in thein vitro culture production method for producing serum-free andmitogen-free interleukin-2 preparations disclosed in the above mentionedprior applications Ser. No. 193,112 and Ser. No. 247,769, thedisclosures of which are incorporated herein, in their entirety, byreference thereto; specifically this invention relates to the use of aroller bottle culture system for improving the yield of interleukin-2.This invention also relates to a further improvement wherein thecultivation is carried out under high oxygen concentration.

There has been an extensive amount of research in recent years regardingthe immunopotentiating effects of various lymphokines, includinginterleukin-2. In July 1980, an International Workshop on Interleukin-2was held in Geisenheim, West-Germany and a symposium publication basedon this meeting was published in Behring Institute Mitteilunger, No. 67,December 1980.

Murine and human Interleukin-2 have been partially purified andcharacterized as described for instance by S. A. Rosenberg, et al "InVitro Growth of Murine T Cells. III. Method for Separation of T CellGrowth Factor (TCGF) From Concanavalin A and Biological Activity of theResulting TCGF," J. of Immunological Methods, 33 (1980), pp. 339-350; J.Watson, et al "T-Cell Growth Factors: Interleukin-2," Immunology Today,December 1980, pp. 113-116; D. Mochizuki, et al, "Biochemical andBiological Characterization of Lymphocyte Regulatory Molecules. IV.Purification of Interleukin 2 from a Murine T Cell Lymphoma," J. ofImmunology, Vol. 125, No. 6, (Dec. 1980), pp. 2579-2583; J. W. Mier andR. C. Gallo "Purification and Some Characteristics of Human T-CellGrowth Factor (TCGF) From PHA-Stimulated Lymphocyte Conditioned Media"

The immunopotentiating effects of lymphokines, especially interleukin-2have been described by several researchers, including, for example,Papermaster, et al "Preliminary Observations on Tumor RegressionsInduced By Local Administration of a Lymphoid Cell Culture SupernatantFraction in Patients With Cutaneous Metastatic Lesions," J. ofImmunology and Immunopathology, Vol. 5, pp. 31-47 (1976); Henny, et al"Interleukin-2 Augments Natural Killer Cell Activity," Nature Vol. 291,May 28, 1981, pp. 335-338; Lotze, et al., "The In Vivo Distribution ofAutologous Human and Murine Lymphoid Cells Grown in T Cell Growth Factor(TCGF): Implications for The Adoptive Immunotherapy of Tumors," J. ofImmunology, Vol. 12, No. 4 (October 1980). The inventors own clinicalstudies have demonstrated the lack of any harmful side effects by the invivo administration of interleukin-2 in tumor patients.

The importance of serum-free tissue culture systems has been recognizedby several authors, for example, see B. W. Needleman and J. M. Weiler,"Human Lymphocyte Transformation Induced by Mitogens And Antigens In ASerum-Free Tissue Culture System," J. of Immunological Methods, 44(1981), pp. 3-14; H. S. Warren and R. G. Pembrey, "A Method For TheProduction And Quantitative Assay Of Human Lymphokine Preparations," J.of Immunological Methods, 41 (1981), pp. 9-21.

Production of a lectin-free murine interleukin-2 preparation isdescribed by P. J. Spiess and S. A. Rosenberg, "A Simplified Method ForThe Production Of Murine T-Cell Growth Factor Free Of Lectin," J. ofImmunological Methods, 42 (1981), pp. 13-222.

However, to date, it has not been possible to produce Interleukin-2 on alarge scale in culture systems nor has there been any successful effortsto produce Interleukin-2 synthetically or by genetic engineering.

Attempts to optimize the yield of Interleukin-2 in vitro cell culturessystems has largely centered on, for example, optimization of type ofand mitogen concentration, cell number and cell type, and other similarfactors. See, for example, Jose M. Alvarez, et al "Human T Cell GrowthFactor I. Optimal Conditions For Its Production," J. of Immunology, Vol.123, No. 3 (Sept. 1979), pp. 977-983. While some degree of success hasbeen achieved by these methods, still greater yields would be highlybeneficial to the study and applications of Interleukin-2, particularlyto long term and commercial use.

In "Regression of Cutaneous Neoplasms Following Delayed-TypeHypersensitivity Challenge Reactions to Microbial Antigens ofLymphokines," by O. A. Holtermann, et al, J. of Medicine, Vol. 6, No. 2,1975, pp. 157,168, the authors report that they produced lymphokinesfrom mitogens sensitized peripheral blood cells which were incubated ona roller drum at 37° C. However, no further details of the incubationstep using the roller drum are reported. Normally, when cultivation onroller drums is used, the drum is rotated at rates of about 1 to 6revolutions per minute.

In conventional culture systems, e.g. flat dish, culture tube, orculture bottle, the cells being cultured tend to accumulate quickly atthe bottom of the respective culture vessel. Cell accumulation in the invitro production of Interleukin-2 (IL-2) appears to result during themitogen (e.g. PHA) stimulation step when the cells tend to become"sticky" and clump together. This cell accumulation results in:

(1) rapid nutritional depletion of the lower layer of the culturemedium;

(2) accumulation of metabolic products and synthesized proteins (some ofwhich may be toxic or suppressive in respect to IL-2 production);

(3) intensive cell-to-cell contact, again possibly resulting insuppression of IL-2 production by specific suppressor cells.

Media conditioned in such "conventional" systems, i.e. where cells areallowed to settle and where no circulation of the medium is provided,require concentration, on the order of 5 to 10-fold, to yield IL-2levels high enough for the in vitro growth of cytotoxic IL-2 dependentT-cells. In fact, some batches require even higher concentration, whileothers show no activity at all.

It can, therefore, be appreciated that IL-2 production in theseconventional culture systems is both generally low and highlyunpredictable. This situation is highly undesireable where it is neededto produce IL-2 on a commercial scale. In addition, the conventionalculture systems, especially the use of flat culture dishes, e.g. Petridishes, require long production times and have high space and volumerequirements to obtain useful yields of IL-2, because of the largenumber of plastic dishes required. Similar space and volume requirementsexist with culture tube systems.

Another economic drawback in the conventional culture systems aspresently practiced for production of interleukin-2 from human sourcesis the high cost of obtaining the peripheral mononuclear blood cells andthe resulting waste of the non-used portions of the starting whole bloodsupply.

It has also been found by the present inventors that in the large scaleproduction of interleukin-2 by in vitro culture systems, in which themedium in the culture vessels form layers, often in excess of severalinches, a substantial barrier to oxygen diffusion (from the air of theair/CO₂ mixture in the incubator) into the liquid media is presented. Infact, liquid layers in excess of 3 to 5 millimeters show somesignificant barrier to oxygen penetration throughout the thickness ofthe liquid conditioning media.

These and other drawbacks are now avoided by the various embodiments ofthe present invention which are all improvements over the static invitro cell culture system described in our copending applications Ser.No. 193,112 and Ser. No. 247,769.

Accordingly, in one aspect, the present invention provides a process inwhich the peripheral mononuclear blood cells (PBL) are packed on aporous support (e.g. nylon wool, sepharose gel, hollow fiber systems,etc.) and the liquid cell culture medium and atmosphere are recirculatedthroughout the incubation and replenished with fresh culture medium bydiafiltration. The recirculation of only the culture fluids tends toprevent depletion of the nutrients in the lower layer of the culturemedium and removes metabolic and synthesized products from the cellenvironment.

In a more preferred embodiment of the invention directed towardprevention of cell clumping or accumulation and its deletorious sideeffects the incubation in both the stimulating and conditioning steps isperformed in a rapidly rotating roller bottle culture system.

The roller culture system avoids all of the above mentioned drawbacksresulting from cell accumulation, including the cell-to-cell contactwhich is not particularly addressed by the first mentioned embodiment.Thus, the rapidly rotating culture bottle system provides:

(1) intensive mixing by the roller action to assure a completelyhomogeneous culture medium during the whole conditioning and stimulatingperiods and thereby prevents local depletion;

(2) dispersion of metabolic products and synthesized proteins throughoutthe whole culture volume, thereby resulting in only a slow and largelyinsignificant buildup of possibly toxic or suppressive productconcentrations;

(3) maintenance of uniform cell suspension thereby preventingsuppression resulting from cell-to-cell contact.

Other advantages of the roller culture system include the capability ofhandling large volumes of culture medium permitting production of up toseveral 1000 liters--on a pilot plant scale--of highly active IL-2conditioned media; relatively low costs due to reusable bottles, lowspace and volume requirements, and handling of large unfractionatedvolumes. In fact, for in vitro growth of IL-2 dependent cytotoxicT-cells, it is often unnecessary to concentrate the conditioned media,although concentration of the conditioned media by only 2 to 5-fold ispreferred.

In another aspect of the invention, especially useful for large scaleproduction, in both static (e.g. flat dish) and dynamic (i.e. rollerbottle) culture systems the yield of IL-2 is improved 5 to 10-fold ormore by carrying out the incubation steps under high oxygenconcentrations, in particular with gaseous mixtures containing fromabout 70 to 95% oxygen, 5 to 15% carbon dioxide, and the remainderprimarily nitrogen. In static culture systems the long diffusiondistances for oxygen in the relatively deep liquid cell culture mediumis compensated for by the higher oxygen concentration resulting inincreased concentrations of IL-2 which are typically 5 times higher thanthe yields obtained in a conventional air/CO₂ gas mixture.

In the roller culture system, the presence of high oxygen concentrationsprovide reliably high yields and oxygen concentrations of IL-2 in theconditioned media allowing the conditioned media to be diluted up to 50times, preferably up to 5 to 10 times, for the in vitro growth ofcytotoxic T-cells. In comparison, IL-2 conditioned media produced inroller culture with a normal air/CO₂ atmosphere have to be concentrated0 to 5-fold to support growth of IL-2 dependent cytotoxic T-cells. Thisis, of course, still a significant improvement over the static culturesystems.

Even in the embodiment using a porous support with packed cells insteadof the roller culture, the higher oxygen concentration providesincreased yields of IL-2 and offers the advantage of a more easilycontrolled oxygen supply by continuously oxygenating and otherwisereconditioning (e.g. pH, nutrients, product removal, etc.) the cell-freecirculating culture medium.

In still another aspect of the invention, applicable to each of theabove described embodiments, the economics of the process aresubstantially improved by using buffy coat cells for the peripheralmononuclear blood cells. Buffy coat cells have the advantage that theyare obtained as a by-product from whole blood which has been treated torecover whole blood plasma and erythrocytes (red blood cells) and do notrequire addition of heparin to prevent blood clotting. Thus, the buffycoat cells which are normally discarded can be recovered atsubstantially no additional cost over the normal production of red bloodcells and whole blood plasma from blood banks. Moreover, the recovery ofleukocytes is higher from buffy-coat cells than from PBL obtained by theprocedures described in our prior applications and which are well knownin the art.

The above and other objects of the invention are achieved in a firstembodiment in which serum-free and mitogen-free interleukin-2 containingsupernatant is produced by (1) stimulating IL-2 producing primary cellswhich have been washed to remove substantially all serum proteins boundto the external cell membrane surface by incubating the washed cells ina liquid tissue culture medium supplemented with serum protein and amitogen in a roller bottle culture system which is caused to rotate at aspeed sufficient to provide a homogeneous culture medium, dispersemetabolic products and synthesized proteins throughout the culturemedium, and maintain a uniform cell suspension in the culture medium;(2) separating and washing the stimulated cells to remove substantiallyall of the serum protein and mitogen; and (3) conditioning the cellsobtained in step (2) by incubating the cells in the presence of aserum-protein-free and mitogen-free liquid tissue culture medium in aroller bottle culture system which is caused to rotate at a speedsufficient to provide a homogeneous culture medium, disperse metabolicproducts and synthesized proteins throughout the culture medium, andmaintain a uniform cell suspension in the culture medium, to therebytransfer interleukin-2 into the liquid phase. Generally, for a bottlehaving a diameter of 4 to 5 inches, a roller speed of at least 20r.p.m., corresponding to about 6 revolutions per minute of the bottleitself, is sufficient to obtain the objectives of the invention. As inour original method the stimulation and conditioning incubation stepsare carried out for periods of about 4 to 8 hours and 18 to 24 hours,respectively.

According to a preferred mode of this embodiment, as described in theabove-mentioned prior applications, the liquid tissue culture mediumfrom which the stimulated cells have been separated are recycled tostimulate subsequent batches of cells, and the conditioned cellsseparated from the tissue culture medium are also recycled forrestimulation.

According to the procedure described in our previous applications, theincubations during stimulation and conditioning are carried out understatic conditions. That is, the cells and liquid tissue culture mediumsupplemented with serum and mitogen in the case of the stimulation stepand the cells in the presence of the serum-free and mitogen-free liquidtissue culture medium in the case of the conditioning step are simplyplaced in a suitable container, such as a Petri-dish, and placed in theincubator where they are incubated at a temperature of about 37° C. withan air/CO₂ -mixture containing 5 to 10% CO₂.

It has now been discovered that when the cells are cultured in thestatic vessel, the interleukin-2 producing cells tend to clump together,thereby reducing the number of available cells for producinginterleukin-2; at the same time the concentration of IL-2 tends to besubstantially higher in the bottom part of the still-standing culturevessel and, therefore, there is a greater tendency for consumption ofthe so-produced interleukin-2 to be adsorbed on cells in the culturevessel carrying receptors for interleukin-2.

In order to avoid the clumping phenomenon and to obtain a more uniformconcentration of interleukin-2 throughout the culture vessel, it wasattempted to utilize a roller culture system, using a conventionalroller culture machine making from 1 to 6 revolutions per minute.However, operating at these conventional speeds did not prevent thecells during the mitogen (e.g. phytohemagglutinin-PHA) stimulation frombecoming "sticky" and clumping together. Using a four inch diameterbottle, it was found that the cells producing lymphokines still clumpedtogether at speeds of rotation as high as 15 r.p.m.

In accordance with the present invention, clumping of the cells in theroller culture vessel is totally or substantially totally eliminated bymodifying the rollers of the roller culture system to make at least 20revolutions per minute (corresponding to about 6 revolutions for a 5inch diameter bottle). The serum and mitogen-free culture supernatantshave a ten-fold and higher increase in yield of interleukin-2 whenproduced by the modification of the previously disclosed method whereinthe incubation during the stimulating and conditioning steps isperformed in a roller culture system which is caused to rotate at atleast 20 r.p.m.

It has also been discovered that even further improvements in yield ofinterleukin-2 in the culture supernatants can be achieved by modifyingthe surface of the roller bottle to remove any irregularities in itssurface. It has been found that such irregularities in the surface ofthe roller bottle, which is typically made from glass material, tend totear up the cells including the interleukin-2 producer cells. Thesmoothing of the roller surface has been successfully achieved byproviding a thin silicone coating on the roller surface. Thus, in theabsence of the siliconization of the roller in the roller culture vesselsome 30 to 50% of the starting peripheral mononuclear blood cells arelost during the PHA stimulation whereas only some 15 to 30% of the cellsare destroyed after siliconization of the roller culture vessel.

In a second embodiment of the invention improved yields of IL-2 areobtained by (1) stimulating IL-2 producing primary cells which have beenwashed to remove substantially all serum proteins bound to the externalcell membrane surface by incubating the cells in a liquid tissue culturemedium supplement with serum protein and mitogen under an oxygen-richatmosphere comprising from about 70 to about 95% oxygen and from about 5to 15% carbon monoxide (the remainder being primarily nitrogen withminor amounts of other inert gases normally found in air); (2)separating and washing the stimulated cells to remove substantially allof the serum protein and mitogen; and (3) conditioning the cellsobtained in step (2) by incubating the cells in the presence of a serumprotein-free and mitogen-free liquid tissue culture medium under anoxygen rich atmosphere as described in step (1).

This embodiment using an oxygen rich atmosphere during the incubation(stimulation and conditioning) steps can be applied to the rollerculture system of the first mentioned embodiment, or to a static, e.g.flat dish, tube, or non-rotating bottle, culture system. Recirculationof both the liquid tissue culture medium and the conditioned cells isalso applicable to this embodiment.

In a preferred mode of carrying out the above described secondembodiment of the invention as applied especially to the firstembodiment the IL-2 producing cells are buffy-coat cells obtained as aby-product during the separation of red blood cells and plasma fromwhole blood by conventional techniques. According to this embodiment thebuffy-coat cells should be substantially fresh, e.g. no more than 2 to 3hours old, when used in the production of interleukin-2.

The invention will now be described in greater detail for a betterunderstanding thereof with the aid of the following non-limitatingexamples.

The roller cultivation apparatus can be any conventional device forrolling bottles containing tissue culture medium and cells. Suchapparatus is widely used in the field of biology, especially in virologyfor the production of viruses. However, these devices normally operateat relatively low speeds, on the order of from about 1 to 6 r.p.m. Thedevice must, therefore, be modified to operate at higher speeds of atleast about 20 r.p.m.

Typically, a roller cultivation apparatus will include at least onepair, and preferably two to four pairs of horizontally disposed parallelrollers spaced apart a short distance sufficient to allow the rollerculture bottle to rest on both rollers of a pair. Rotation of one orboth rollers of a pair causes rotation of the bottle in contacttherewith. The direction of rotation is unimportant.

One arrangement of the apparatus which has been found useful includesthree parallel rollers which can accomodate two bottles. All threerollers can be rotated or only the outer two can be driven and themiddle roller can be freely rotating. It is also possible for only themiddle roller to be driven and the outer two freely rotating. Two ormore sets of the three rollers can be provided in stacked relationshipso long as sufficient clearance is provided between the rows toaccommodate the diameter of the bottle.

As stated above, these roller culture devices are normally designed toimpart a rotation to the rollers of only 1 to 6 r.p.m.

Studies by the inventors have shown that at these speeds clumping of thesticky cells cannot be avoided. By modifying the apparatus to produceroller speeds as high as 15 r.p.m. it was still not possible to overcomethe clumping phenomenon. However, by further modifying the apparatus toproduce roller speeds of at least 20 r.p.m. it becomes possible toprevent the sticky interleukin-2 producing cells from formingagglomerates with the attendant disadvantages thereof.

The upper limit of the roller speed is not particularly critical butshould not be so high as to cause rupture or breakdown of the cells.Usually roller speeds up to about 50 r.p.m. are satisfactory and noparticular advantage is obtained by operating at higher speeds.Generally, roller speeds on the order of about 20 to 50 r.p.m.preferably 22 to 40 r.p.m., which correspond, for roller bottles rangingin diameter from 4 to 5 inches and containing from about 50 to 200 ml ofculture medium, to roller bottle rotations of from about 5 to about 20r.p.m., preferably from about 5 to 15 r.p.m., are sufficient to avoidclumping of the sticky cells.

For larger or smaller size roller bottles, larger or smallerconcentrations of cells and amounts of culture medium, different amountsof rolling friction between the rollers and roller bottles, differentdiameters of the rollers, etc., the minimum rotation speed of therollers to effect sufficient mixing to assure complete homogeniety ofthe culture medium, dispersion of metabolic and synthesized products,and maintenance of uniform cell suspension, can be readily determined bysimple experimentation.

It is preferred, especially when the roller bottle vessels are to berepeatedly used for stimulating and conditioning the cells, to avoidformation of surface irregularities. For example, the roller bottles aremade of glass. After several washings, the surfaces of these bottlesbecome rough and irregular tending to tear the cells up. One means forpreventing the roughening of the glass surface is to coat it with asilicon solution. Such procedure is known in the art.

For example, one suitable siliconizing solution is commerciallyavailable under the tradename "Siliconlosung Serva," article number35130 IIIa, available from Serva, Heidelberg, Federal Republic ofGermany. A small volume of silicone solution is poured into the rollerbottle. When the inner surface has been completely covered by silicone,excess solution is decanted off. The bottle is dried at room temperaturewith the nozzle downwards. The remaining film of silicone is now burnedinto the glass by heating the bottle for 1 hour at 100°-150° C. Thetreated bottle has a very smooth surface which is hydrophobic and it maybe used for 5 to 10 times before a new siliconization procedure will benecessary. The siliconization treatment is important to reduce loss ofcells during stimulation and conditioning.

The cultivation process using rolling bottles may be essentially thesame in all other aspects as in the process described in our priorapplications, e.g. Ser. No. 247,769. Thus, the IL-2 producing cells maybe any normal (i.e. non-cancer or non-tumor) primary cell system such asperipheral mononuclear blood lymphocytes which are obtained and washedas previously described. The cell donor may be sheep, pig, cattle orhuman. The temperature and atmosphere may also be those previously andconventionally used, e.g. 37° C. and an air/5-15% CO₂ atmosphere.

However, for best results in terms of economy and yields ofinterleukin-2, particularly in applications for human use, the IL-2producing cells can be buffy coat cells and the atmosphere should beoxygen enriched, for example 70-95% O₂, preferably 80-95% O₂ (percent byvolume), with about 5 to 15%, preferably 5 to 10% carbon dioxide (CO₂)as a buffer, and the remainder is substantially nitrogen, with a minoramount of inert gases, such as argon, normally found in air.

EXAMPLE 1

As a specific example of the roller bottle culture process peripheralmononuclear blood cells are first obtained as previously described.Briefly, human blood obtained from a blood bank is mixed with heparin, aclotting inhibitor. The blood is layered into Ficoll-hypaquemixture-containing sterile tubes which are then centrifuged. Theresulting layer of peripheral mononuclear blood cells formed above theFicoll-hypaque layer in each tube is drawn off carefully with a pipet.The cells are then pooled in a clean tube and diluted with, for exampleRPMI 1640. The tube is centrifuged at speeds which do not rupture thecells and the supernatant is discarded. The cells are then washedrepeatedly with fresh portions of RPMI solutions in the same manner. Theresulting washed cell pellet containing about 500-1000 million cells isdiluted with about 5 to 10 ml RPMI 1640 solution.

The cells may then be seeded out at a density of about 3×10⁶ /ml in atissue culture medium, e.g. RPMI 1640 containing about 4 micrograms ofphytohemagglutinin (PHA) supplemented with 10-15% blood serum. In placeof the blood serum, the plasmatic human interleukin-2 inducing protein(PHILIP) described in the copending U.S. application Ser. No. 255,251,filed Apr. 17, 1981, the disclosure of which is incorporated herein byreference thereto, may be used.

At this point, the cell- and lectin-containing culture medium istransferred to roller culture bottles. Bottles ranging in diameter offrom 4 to 5 inches are used and each bottle is filled with from 50 to200 ml of culture medium. The roller bottles, after being filled with anair/CO₂ (5-10%) mixture, are placed in an incubator equipped with theroller apparatus. The temperature in the incubator is maintained atabout 37° C. The rollers are rotated at about 25 r.p.m. (a roller bottlespeed of about 6 r.p.m.) and the cells are incubated for about 4 to 8hours, preferably about 4 hours.

Thereafter, the bottles are removed from the incubator, and thestimulated cells containing culture medium is decanted into centrifugetubes, and the cells sedimented and washed three times in theconventional manner as previously described, and then seeded out intofresh culture medium, again at a cell density of about 3,000,000/ml. Inthis case, the cell containing culture medium, which is not supplementedwith PHA or serum proteins, is again transferred into the roller bottlesin 50 to 200 ml portions and incubated under the same conditions used inthe stimulation step except that the time of the conditioning incubationis increased to about 18 to 24 hours, preferably about 20 hours.

Thereafter, the medium is again transferred from the roller bottles tocentrifuge tubes and processed as in our previous application (seeExample 1 of Ser. No. 247,769). The cells can be reutilized insubsequent PHA restimulation and reconditioning for at least 5additional cycles.

EXAMPLE 2

Example 1 is repeated except that the incubation in the stimulation andconditioning steps is performed under an atmosphere containing 90% O₂,5% CO₂ and 5% N₂.

The interleukin-2 activity of the conditioned culture supernatants fromthe roller bottle plus air/CO₂ mixture, from the roller bottle plus 90%O₂, and from conventional PHA stimulation in 50 ml Falcon blue capplastic tubes with normal atmospheric oxygen (air)+10% CO₂) (ComparativeExample 1) were tested (three replications each) and the results areshown in the follow table:

    ______________________________________                                        IL-2 ACTIVITY IN                                                              CONDITIONED CULTURE SUPERNATANTS                                                       Preparation Procedure                                                         Plastic Tubes                                                                           Roller bottles                                                                           Roller bottles                                           + air/CO.sub.2                                                                          + air/CO.sub.2                                                                           + 90% O.sub.2                                            (Comp. Ex 1)                                                                            (Ex. 1)    (Ex. 2)                                         ______________________________________                                        cells per well                                                                            95 ± 12*                                                                              *          243 ± 27*                                × 10.sup.-3                                                                        (1:4)                  (1:8)                                       (dilution factor)                                                             ______________________________________                                         *Average of 3 experiments                                                

COMPARATIVE EXAMPLES 2-3

Examples 1 and 2 are repeated except that the rollers were rotated atspeeds of only about 15 r.p.m. The results are substantially the same asComparative Example 1 and substantial cell clumping is observed.

EXAMPLE 3

Example 2 is repeated except that buffy coat cells are used in place ofthe PBL cells. The buffy coat cells are obtained as follows: Blood iscollected in a vessel containing a preservative for the erythrocytes,e.g. ACD (Adenine, citrate, dextrose) which is a standard solution forpreserving erythrocytes. The use of heparin, which is needed for theinterleukin-2 production in whole blood conserves is not necessary.After collection, the fresh blood specimen is centrifuged at 1000 to1500×G for 20 minutes. The erythrocytes will sediment and the leukocyteswill form a thin layer (buffy-coat) on the erythrocyte sediment.Thereafter, the buffy-coat is aspirated off the erythrocyte layer andused for the production of interleukin-2. The leuko- anderythrocyte-free plasma can be collected and used, for example, forindustrial purposes or for the infusion as whole plasma into patients.The sedimented erythrocytes can be used for clinical purposes (e.g.blood transfusion) as usual. Most blood banks prepareerthrocyte-concentrates in the way described above only with thedifference that the buffy-coat is not collected. The leukocytes aregenerally considered worthless. The blood conserve usually stands in arefrigerator for some day or weeks before it is used. During this time,the leukocytes will die. Using these buffy-coat cells, it is possible toget raw material for the interleukin-2 production almost withoutadditional costs for the blood bank.

When the leukocytes are now processed for IL-b 2 production, they areseparated from contaminating erythrocytes and thrombocytes by aFicoll-hypaque gradient in exactly the same manner as previouslydescribed. The cell number recovered will usually be about 20% higherthan the cell number recovered from a heparin-conserve. It is veryimportant that the buffy-coat cells are absolutely fresh, when they arestimulated for the IL-2 production. They should not be older than 2 to 3hours. It has been found, quite unexpectedly that the use of buffy-coathas one limitation: If one tries to produce IL-2 in buffy-coats undernormal oxygen concentration, no sufficient production will be found. Oneprerequisite, therefore, is the use of an oxygen rich atmosphere in thesame manner previously described. The result obtained are substantiallythe same as in Example 2.

COMPARATIVE EXAMPLE 4

When example 2 is repeated except that buffy-coat cells are used inplace of the PBL cells, substantially no IL-2 production is observed.This example shows that buffy coat cells require high oxygenconcentrations during incubation, although the reason for this has notbeen fully elucidated.

The interleukin-2 containing supernatants obtained by the improvedprocedures of this invention, after filtering e.g. on an Amicon YM-10filter as described in our prior applications, is a highly purifiedproduct. Product purity of the interleukin-2 containing conditionedsupernatant, can be demonstrated by comparison of the biologicalactivity with the protein content. Biological activity of suchinterleukin-2 containing preparations can be detected in materialdiluted up to 50 times before filtering on an Amicon YM-10 filter. Afterfiltering, the biological activity can be multiplied by a factor whichis identical with the reduction of the sample volume during thefiltration process. An interleukin-2 containing supernatant which beforefiltering has a biological activity which is detectable in 50 folddilution and which is concentrated by filtration to a tenth of itsoriginal volume will after the filtration have a biological activitydetectable in a 1:500 dilution. The protein content of the filteredsupernatants usually ranges from 5 to 20 μg/ml, due to the inherentdifferencies in cell loss from cell batch to cell batch. Also, the cellsof each donor have inherently different capacities to produce IL-2 bymitogen stimulation.

Accordingly, it is recognized that the present invention providessignificant improvements in yield and in the overall economy of theinventor's original procedure for the in vitro production of high purityserum-free and mitogen- (e.g. lectin-) free interleukin-2 containingconditioned supernatants. These supernatants are useful in the treatmentof patients having depressed natural killer cell function or havingdepressed T cell function, in the diagnosis and treatment of immunedeficiency in tumor patients, and in general for any of the utilitiespreviously disclosed in application Ser. No. 247,769, or by otherauthors.

What we claim is:
 1. In a process for producing a serum-free and mitogen-free interleukin-2 containing supernatant by the steps of stimulating IL-2 producing cells by incubating the cells in a liquid tissue culture medium supplemented with serum protein and mitogen; separating and washing the stimulated cells to remove substantially all of the serum protein and mitogen; and, conditioning the washed cells by incubating the cells in the presence of fresh liquid tissue culture medium in the absence of serum protein and mitogen to thereby transfer the interleukin-2 into the liquid phase; the improvement comprising,carrying out the incubation steps under an oxygen rich atmosphere comprising at least 70% oxygen and at least 5% carbon dioxide.
 2. The process of claim 1 wherein the oxygen rich atmosphere comprises 80 to 95% O₂, 5-10% CO₂ and the remainder is substantially nitrogen.
 3. The process of claim 1 or claim 2 wherein the IL-2 producing cells are buffy-coat cells obtained from a human donor. 